The present invention relates to monoclonal antibodies reacting with certain epitopes of recombinant bovine prion protein, native and denatured normal or disease-specific prion proteins in soluble or insoluble state, stable hybridoma cell lines producing these monoclonal antibodies, recombinant expression vectors for the expression of recombinant bovine prion protein, purified recombinant bovine prion protein, a test kit for the diagnosis of prion diseases, diagnostic methods for the immunological detection of prion diseases, pharmaceutical preparations for the prevention and therapy of prion diseases, a method for clearing biological material from infectious prion proteins, and methods for the production of these materials.
Abbreviations used hereinbefore and hereinafter are the following:
Prion diseases are transmissible neurodegenerative diseases of the central nervous system (for review see Prusiner, 1991). They can be transmitted, inherited or occur sporadically and are observed in animals (e.g. bovine spongiform encephalopathy [BSE] in cattle, scrapie in sheep) as well as in humans (Creutzfeldt-Jakob disease, Gerstmann-Strxc3xa4ussler-Scheinker syndrome, Fatal Familial Insomnia, Kuru). Prion diseases have a characteristically long incubation period and, with the onset of clinical symptoms, lead to ataxia, dementia, psychiatric disturbances and sleeplessness before inevitable death occurs. Neuropathological changes include vacuolar degeneration of brain tissue, astrogliosis and amyloid plaque formation. In the infected subjects, neither a systemic immune response, nor an obvious specific immune response like antibody production of PrP has been observed (Kasper et al., 1982; Garfin et al., 1978) however, some unspecific activation of immune cells in the brain was reported (Williams et al., 1995; Williams et al., 1994).
The infectious agent appears to exist in a variety of strains, which cause distinct incubation times and histopathology (Bruce et al., 1994; Hecker et al., 1992). Transmission of prion diseases is possible between species and most easily within the same species (Prusiner, 1991).
The infectious agent, the prion, is associated with a disease-specific protein, PrPSc, that is an abnormal isoform of a host protein, PrPC (Oesch et al., 1985, Basler et al., 1986). Both, PrPSc and PrPC, have an apparent molecular weight of 33-35 kDa on SDS-polyacrylamide gels. They have the same amino acid sequence and are glycosylated at two asparagine residues (Oesch et al., 1985) After proteinase K treatment, PrPSc is shortened to a characteristic 27-30 kDa fragment while PrPC is fully digested (Bolton et al., 1982, Oesch et al., 1985), this led to the conclusion that the disease-specific isoform PrPSc is partially protease resistant while the normal host isoform PrPC is not.
Studies on the synthesis and localization of the two PrP isoforms in cultured cells have shown that PrPC is attached to the cell surface by a glycosyl phosphatidylinositol (GPI) anchor while PrPSc accumulates intracellularly within cytoplasmic vesicles (Stahl et al., 1987). Another difference between PrPC and PrPSc is reflected in their three-dimensional structure PrPSc has less alpha helical secondary structures and increased beta sheet content as compared to PrPC (Pan et al., 1993). So far, no chemical differences between the two isoforms have been observed (Stahl et al., 1993). In summary, PrPSc and PrPC have the same amino acid sequence but a different folding. The misfolded prion protein is associated with infectivity and neurotoxicity.
The infectious agent is inactivated by treatments which denature proteins while reagents destroying nucleic acids have no effect (Diener et al., 1982; Alper et al., 1978). In addition, no single nucleic acid capable for coding a protein has been purified until date (Riesner et al., 1993). This has lead to the hypothesis that PrPSc itself might comprise the infectious particle (Griffith, 1967; Prusiner, 1982). According to this hypothesis, replication of infectivity is achieved by the replication of the pathogenic conformation. It is supposed that infectious PrPSc molecules convert the normal host protein PrPC to the PrPSc conformation (Cohen et al., 1994). Conversion of PrPC to PrPSc was claimed to have been achieved in vitro thereby mimicking species and strain characteristics comparable to the conversion dynamics in vivo (Kocisko et al., 1994; Bessen et al., 1995). However, these in vitro converted PrPSc molecules have, to date, not shown to be infectious.
The function of the normal host protein, PrPC, is unknown. Mice devoid of PrPC are viable and show no obvious signs of neurological and physical impairment (Bueler et al., 1992). In addition, these mice are not susceptible to infection with prions, underlining the central importance of PrP in the replication of infectivity and/or pathology of these diseases (Bueler et al., 1993; Prusiner et al., 1993). More subtle investigations of PrP knockout mice revealed impaired synaptic function (Collinge et al., 1994) and altered sleep regulation (Tobler et al., 1996). However, a molecular function of PrPC could not be deduced from these findings.
Prion diseases have gained public interest with the appearance of BSE in the early eighties in Great Britain (Hope et al., 1988); for review see (Wells and Wilesmith, 1995). The disease is supposed to have been transmitted by feeding prion-contaminated meat and bone meal to cattle. It is thought that BSE prions originated from scrapie-diseased sheep by crossing the species barrier from sheep to cattle. BSE has caused an epidemic or considerable importance for both, public health and cattle-dependence economy. Remarkably, no diagnostic method suitable for mass screening of infected tissues of cattle has been developed to date.
Initial diagnosis for prion diseases classically relies on the appearance of clinical symptoms. A definitive diagnosis is made by the observation of neuropathological changes in the medulla oblongata. In few cases, BSE has been shown to be transmissible to other cattle, sheep, pigs and mice. Modern diagnosis additionally uses immunological detection of PrPSc in brain sections. Since PrPSc can be detected in the CNS after half of the incubation time in experimentally infected laboratory animals (Jendroska et al., 1991; Hecker et al., 1992), it may serve as an early marker of infection. Hence, specific and sensitive detection of PrPSc allows the identification of infected animals at a subclinical stage and will help to reduce possible human health risks. By autumn 1996, the BSE epidemic has killed over 160,000 cows in Great Britain alone. In the absence of a diagnostic test, only cattle with clinical symptoms were sorted out from being further processed, allowing a great number of BSE-infected cattle to enter the human food chain (Anderson et al., 1996). This lead to the suspicion that the appearance of a new variant of Creutzfeld-Jakob disease in Great Britain was caused by transmission of BSE to humans (Will et al., 1996; Colinge et al., 1996). A sensitive detection method for bovine PrPSc will allow the identification and removal of subclinical BSE-cases from the human food chain.
Oesch et al., (1994) have used a procedure that allows to quantitate the disease-specific isoform of PrP in hamsters. The procedure is based on an ELIFA (enzyme-linked immuno-filtration assay), and is adapted to the particularities of the prion protein, especially the poor solubility of the disease-specific isoform that has made application of conventional ELISA techniques difficult. This procedure (described in detail below) allows for testing of thousands of samples and is thus appropriate for routine screening of animals and humans for prion diseases.
Tagliavini et al (WO 93/23432) describe a method for detecting soluble prion polypeptides. The drawback of this method is that the inventors claim to detect prion poypeptides that are soluble in vivo, however, it is known since a long time that the disease-associated prion protein PrPSc is insoluble in vivo. State of the art is that insoluble PrPSc has to be solubilized in vitro to be detected by immunological methods. Tagliavini et al state (page 3, row 31) xe2x80x9c . . . such truncated scrapie proteins have not been found to exist in vivo in substantially soluble formxe2x80x9d. Furthermore, the inventors give an example wherein they show soluble prion polypeptide fragments in the cerebrospinal fluid (CSF) of patients that do not suffer of the human prion disease CJD but of other unrelated diseases. However, the inventors do not show in vivo soluble protease-resistant prion polypeptides which would prove their hypothesis about the existence of disease-specific prion polypeptides in CSF. In addition, to show prion polypeptides in CSF they use an immunoblot (Western blot); this technique is not appropriate to detect naturally occurring soluble prion polypeptides, since the immunoblot technique requires solubilization of proteins in vitro prior to gel electrophoresis. This procedure would then solubilize even insoluble prion polypeptides that would be suspended in CSF.
Major shortcomings for the immunological detection of PrP have been the unavailability of excellent antibodies able to detect the native disease-specific prion protein (Kascak et al., 1987; Barry and Prusiner, 1986; Takahashi et al., 1986; Barry et al., 1986). In particular, native PrPSc was invisible to antibodies (Serban et al., 1990). Furthermore, no monoclonal antibodies recognizing the bovine PrP were available. The reason for the difficulties in raising monoclonal as well as polyclonal antibodies is the highly conserved amino acid sequence of PrP in mammals which apparently prevents an antibody response against most epitopes.
Kascsak et al., (1987) describe the monoclonal antibody 265K3F4 produced by hybridoma cell line ATCC HB 9222 directed against scrapie-associated fibril proteins. The drawback of this method is that by immunizing wild-type mice with PrP, due to self-tolerance, an antigenic reaction against many epitopes is suppressed. The inventors immunized wild-type mice with purified scrapie-associated fibrils (SAF); SAF are multimeric complexes consisting of PrPSc that are purified by a ultracentrifugation. The inventors describe an antibody, termed 3F4, that binds only to hamster and humans PrP. Furtheron, the antigen has to be denatured either by formic acid or SDS to be detected. It is stated (Kascsak et al., 1987) that the 3F4 antibody binds to undenatured SAF 10-fold weaker than to formic acid-denatured SAF. However, the 3F4 antibody does not distinguish between PrPC and PrPSc.
Williamson et al. (1996) have tried to circumvent the lack of an immune response to a highly conserved protein by immunizing transgenic mice lacking PrP (PrP0/0-mice) with PrP, however, without success. These authors state that after immunizing PrP0/0-mice with PrP, killing these mice for hybridoma production has repeatedly yielded hybridoma cells that failed to secrete anti-PrP antibodies beyond a period of 48 hxe2x80x3. They presume that during the 48 hours after the fusion anti-PrP antibody-secreting clones either are suppressed to secrete further antibodies or die because of an interaction of the secreted antibodies with cell-resident PrP. Williamson et al tried to circumvent this problem by isolating antibody-cording RNA and constructing recombinant antibodies by the phage display technique. They obtained several recombinant antibodies which bind to non-denatured mouse prion rods (PrPSc) in the ELISA technique, however, much weaker than to denatured rods and only if substantial amounts of rods were bound to the wells (0.2 xcexcg/well incubated with 5 xcexcg/ml antibody). However, these recombinant antibodies do not detect native PrPSc in non-denatured histoblots. Thus, the necessity of purifying PrPSc before antibody detection complicates the use of their immunological detection method.
Krasemannm et al. (1996) have made monoclonal antibodies by means of immunizing PrP0/0-mice. After DNA-immunization by injecting the DNA coding for the human prion protein directly into a regenerating muscle the mice were subsequently boosted with Semliki Forest Virus particles containing recombinant human prion protein. The authors present hybridoma cell lines producing monoclonal antibodies that bind to the native and denatured normal human prion protein. The binding of these antibodies to the native or denatured disease-specific prion protein, however, is not demonstrated. Furtheron, the obtained antibodies bind to a peptide ELISA system, however an ELISA to normal or disease-specific prion protein is not shown.
We are now the first to show that immunization of PrP knockout mice with highly purified recombinant PrP followed by fusion of splenocytes from these mice with myeloma cells resulted in hybridoma cell lines that secrete highly specific antibodies to both PrP isoforms (PrPC and PrPSc) in their native as well as denatured state. On the basis of these antibodies, highly specific immunological testing for prion disease was developed.
It is an object of the present invention to overcome the drawbacks and failures of prior art and to provide monoclonal antibodies from stable hybridoma cell lines which can be used in the diagnosis and therapy of prion diseases.
Surprisingly the drawbacks of the prior art can be overcome by immunization of PrP0/0 knockout mice with highly purified recombinant PrP followed by fusion of splenocytes from these mice with myeloma cells. The resulting hybridoma cell lines are surprisingly stable and secrete highly specific antibodies to both PrP isoforms (PrPC and PrPSc) in their native as well as denatured state. The obtained antibodies are very useful for the development of highly specific immunological tests for prion diseases and other purposes.
The present invention concerns a monoclonal antibody or a fragment thereof capable of specifically binding to recombinant bovine prion protein, and native and denatured normal PrPC or disease-specific prion protein PrPSc in an antigen-antibody complex.
The present invention concerns further an antibody or a fragment thereof capable of specifically binding to the binding region (idiotype) of said antibody.
The present invention concerns further a hybridoma cell line capable of producing a monoclonal antibody capable of specifically binding to recombinant bovine prion protein, and native and denatured normal PrPC or disease-specific prion protein PrPSc in an antigen-antibody complex.
The present invention concerns further a recombinant expression vector for the expression of recombinant bovine prion protein.
The present invention concerns further a highly purified recombinant bovine prion protein, which may be in reduced or oxidized form.
The present invention concerns further a method for the production of an antibody as mentioned above, comprising culturing a hybridoma cell line as mentioned above and isolating the monoclonal antibody from the supernatant.
The present invention concerns further a method for the production of a hybridoma cell line as mentioned above, comprising administering to PrP0/0 mice (knockout mice without a functional PrP gene) an immunizing amount of recombinant prion protein as mentioned above, removing the spleen from the immunized mice, recovering sphenocytes therefrom, fusing the latter with P3X63Ag8U.1 hybridoma cells ATCC CRL 1597, growing the cells in a selection medium, screening the cells with recombinant PrP and isolating the positive cells.
The present invention concerns further a method for the production of an expression vector as mentioned above, comprising amplifying DNA from bovine genomic DNA coding for PrP by means of N- and C-terminal primers, and inserting the amplified DNA coding for PrP in the correct reading frame into an expression vector.
The present invention concerns further a method for the production of recombinant bovine prion protein comprising culturing microorganisms or cell lines with an expression vector as mentioned above in an appropriate culture medium and isolating and purifying the recombinant protein.
The present invention concerns further a test kit for the diagnosis of prion diseases.
The present invention concerns further an immunological detection procedure for the diagnosis of disease-specific prion proteins.
The present invention concerns further a pharmaceutical preparation for the therapy and prevention of prion diseases comprising a monoclonal antibody as mentioned above and pharmaceutical carrier.
The present invention concerns further a method for the therapy or prevention of prion diseases comprising administering to a patient suffering from such disease or being likely to becoming a victim of this disease a therapeutical or preventive amount of a monoclonal antibody as mentioned above.
The present invention concerns further a method for clearing biological material from prions comprising treating said material with a monoclonal antibody as mentioned above.